1. State of the Art
It is possible to clone DNA inserts into vectors without ever select for recombinants and instead spend time in identification by hybridisation with radio-labelled probes, screen by restriction of small-scale preparations of plasmids or screening based on the inactivation of the alpha complementation in presence of X-Gal (blue/white screening).
These approaches used for more than one decade are not adapted to large scale cloning projects coming downstream complete genomes sequencing programs. The sequence information available has increased tremendously and will increase further in the future.
In order to assess the biological function of a coding sequence which has been identified, its corresponding gene in the genome of an organism must be specifically mutated (deleted or modified) (for instance in a knock-out mouse), which allows the study of phenotype(s) related to the mutation introduced. The specificity of the 5 mutation is given by a targeting vector (constructed in E. coli) containing homologous recombination arms.
If the development of sequencing technologies has permitted to increase tremendously the number of sequenced genes, cloning and sub-cloning genes at large scale in order to have recombinant clones and get information about the function of the corresponding genes is still a limiting step to functional genomic programs.
Cloning and sub-cloning each gene represent the bottleneck of the “functional genomic” programs. Therefore, new cloning approaches allowing speeding up these processes are required.
Because the identification of recombinants is a limiting step, it is clear that the positive selection of recombinants is required to move from traditional approaches to high throughput cloning allowing working with thousands of genes.
Cloning vectors permitting direct selection (positive selection) of recombinant strains have been proposed (for example, see Pierce et al. 1992; Kuhn et al. 1986). However, most cloning vectors present the following drawbacks: (i) they cannot be used to incorporate large nucleotide fragments, (ii) they are not easy to manipulate and (iv) they cannot be produced by a micro-organism in a large number of copies without bringing about the death of said micro-organism.
Furthermore, traditional restriction and ligase reactions can be replaced by site-specific recombination and recombinants be selected by the replacement of the ccdB gene (a member of the poison/antidote gene family) by the insert of interest (U.S. Pat. Nos. 5,910,438, 6,180,407 and International Patent WO 99/58652).
The main advantages of the ccdB-containing vectors over the other positive selection systems are i) the small size of their selective gene (ccdB: 303 bp), ii) the fact that the vector can be amplified in a host harbouring a mutation that confers total resistance to the CcdB poison (gyrA462 resistant strain; Bernard and Couturier, 1992). Since E. coli is the host used for most molecular cloning strategies, it is important to develop new systems which can enrich and widen the range of cloning possibilities. The positive selection technology using ccdB has been used to derive new vectors adapted to peculiar purposes: PCR cloning vectors (Gabant et al. 1997), vectors adapted for bacterial genetics (Gabant et al. 1998), and recently a kid gene belonging to the CcdB family has been used to design new cloning vectors (Gabant et al. 2000 and WO 01/46444).
Another example of the use of the CcdB gene is given by U.S. Pat. No. 5,888,732. This document discloses the general principle of the cloning method known as “the Gateway system.” In this method, the traditional restriction and ligase reactions are replaced by site-specific recombination sites and the recombinants are selected by inactivation (by deletion) of the ccdB gene by the gene of interest. This method allows rapid and efficient transfer of all the genes from an organism from one vector to different vectors (i.e. expression vectors) by automatic sub-cloning. The resulting sub-clones maintain the orientation as well as the reading frame allowing translation fusions (for a general overview, see Hartley et al. 2000).
However, said techniques are based upon counterselectable genes requiring, the use of rpsl, tetR, sacB or ccdB counterselectable genes, which can generate a seamless second round product that carries no “scar” from the first round of recombination.
Counterselection (selection for the inactivation or deletion of a toxic gene) is typically less efficient than positive selection (acquisition of a new property) as the intended recombination is only one of the several solutions for counterselection pressure. Any mutational event that ablates expression of the counterselectable genes, will also grow under counterselection pressure.
Thus for rare genetic events (which frequency can be compared to the mutational inactivation of the counterselectable gene), candidates from second-round counter-selection strategies, need to be screened to find the intended recombination event.
In practice, it seems that the ratio of the intended to unwanted products varies widely (from <1% to 15%-85%) for reasons that are still undefined (Muyrers G. P. P., Trends in Biochemical Sciences, Vol. 26, no. 5, p. 325-331, 2001).